Unravelling geospatial distribution and genetic diversity of greenhouse whitefly, Trialeurodes vaporariorum (Westwood) from Himalayan Region

The Greenhouse whitefly (GWF), Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae), is a destructive pest that affects protected cultivation worldwide. The Indian Himalayan region is particularly vulnerable to GWF introduction, invasion, and spread due to the expansion of protected cultivation and climate change. In this study, we collected 32 naturally occurring GWF populations, mainly from the Uttarakhand state in the Indian Himalayan region, to investigate the distribution pattern and genetic diversity of T. vaporariorum. Our sampling was representative of the region's vegetation diversity and geographical location, and we collected samples from multiple sites within each locality to account for local variations. The mtCOI gene was used to accurately detect and identify GWF and to sequence haplotypes prevalent in the Uttarakhand state. The maximum likelihood method used for phylogenetic studies revealed that all 32 whitefly samples in this study belonged to T. vaporariorum and were prevalent in all the collected localities. Our population genetic study using mtCOI showed variation within T. vaporariorum populations, with 20 distinct haplotypes present. Notably, haplotype 2 (H2) was the most dominant haplotype among the sampled populations. These results provide fundamental knowledge for understanding the geographical distribution and ecology of T. vaporariorum in the Uttarakhand state of the Indian Himalayan region. The discovery of geospatial and genetic diversity of GWF in the Himalayan region underscores the importance of pest alertness, research prioritization, and the development of sustainable management strategies to protect crops.

www.nature.com/scientificreports/ from foliage, flower, seeds, secreting honey dew, which attracts the development of soot molds fungi 7 . These molds affect photosynthesis in leaves, leading to significant economic loss to the crops 9 . From India, GWF was first recorded from Nilgiri hills of Tamilnadu, southern state of India 10 and successively has been reported in other parts of the country [11][12][13][14] . The increase in area under protected cultivation and climate change in the Uttarakhand states of Indian Himalayan region are critical factors in vulnerability to introduction, invasion, and emergence of GWF in new areas. Therefore, updating the status of GWF, mapping the geospatial distribution pattern, and intraspecific genetic diversity of the burgeoning population is obligatory to ensure timely mitigation of insect pests through suitable pest management operations. Additionally, keeping an eye on GWF springing into new regions is key in sending alertness to the farming community, safeguarding crops, and ensuring maximum returns to farmers. However, the geospatial distribution and genetic diversity for GWF is unknown from the Uttarakhand states of Indian Himalayan region. Moreover, identification of genetic variation in populations of GWF is a key objective to formulate any management plan and pest management decision making.
Traditional identification of GWF is based on morphological traits, which has several drawbacks, including reliance on expert taxonomists, time-consuming, low resolution in presence of conspecific character, presence of cryptic population, and ergonomic issues [15][16][17] . DNA barcoding-based species identification utilizing mitochondrial cytochrome C oxidase subunit I (mtCOI) offers rapid, reliable detection and study of genetic variation among GWF 18 . Therefore, in this study, we aimed to use mtCOI gene markers to unravel the distribution pattern and genetic diversity among GWF (T. vaporariorum) for monitoring, tackling the pestilence to protect the protected cultivations.
Despite a few genetic diversity and distribution studies on GWF from India 19,20 , no populations and genetic diversity study has been conducted for Uttarakhand states, where protected cultivation is an important agricultural enterprise on a large scale. Therefore, we conducted extensive geographical sampling of GWF from protected cultivations, collecting 32 naturally occurring GWF populations representing Uttarakhand states of India. We aimed to identify the mtCOI gene sequence variation-based haplotypes most commonly representing in the sampled geographical region to formulate any management plan and pest management decision making.

Results
A total of 32 T. vaporariorum whitefly samples were collected from commercial host crops including tomato, capsicum, french bean, brinjal, cauliflower, snake guard, okra, bottle guard, garden pea and ornamentals like salvia, marigold. These crops provided a significant food source and habitat for the greenhouse whitefly (GWF) population in the study area. These crops were widely grown in open and protected structures and were heavily infested by GWF. While the chosen host crops may not be exhaustive in terms of their contribution to the genetic variation of GWF, they were important sources for the pest species. The extensive geographical sampling of GWF populations from protected cultivations conducted in this study aimed to capture a wide range of genetic diversity and distribution patterns among GWF in the Indian Himalayan region. The combination of the chosen host crops and the extensive geographical sampling approach was expected to provide a comprehensive understanding of the genetic diversity and distribution patterns of GWF in the study area.
Most of the samples were collected from Uttarakhand and three samples from Himachal Pradesh, one each from Delhi and Tripura. The partial mtCOI gene were successfully amplified and sequenced for molecular identification and phylogeny estimation. The identity of T. vaporariorum in collected samples was supported by morphological and molecular diagnostic technique. The result suggests the frequent occurrence of T. vaporariorum in the surveyed locality including open and protected structures on range of host crops. The surveys were conducted for the first time in few districts like Chamoli, Dehradun, Nainital and Pithoragarh of Uttarakhand state and greenhouse whitefly infestation was recorded for the first time in commercial crops like Tomato, Capsicum, French bean, Brinjal and Cauliflower.
The genetic diversity based on mtCOI gene sequences of collected individuals have been submitted in Gen-Bank database citing the information of locality, host range, geospatial data etc. and the accession numbers have been received and summarized in Table 1. The phylogenetic analysis of 32 samples from various agroclimatic regions of the present study were combined by retrieving some available T. vaporariorum accessions viz., AY521265, NC006280, LN614547 to exploit as ingroups whereas the Genbank accessions viz., KU761949 Aleurocanthus camelliae, NC029155 Aleurocanthus spiniferus, AY572538 Aleurochiton aceris, NC005939 Aleurodicus dugesii, MT880225 Aleyrodes shizuokensis, KR819174 Bemisia afer, MH205754 Bemisia tabaci, AY572539 Neomaskellia andropogonis, NC006292 Tetraleurodes acacia were used as outgroups. Phylogenetic studies based on maximum likelihood method reveals that all the 32 whitefly samples from the study belonged to T. vaporariorum and it is found prevalent in all the collected localities (Figs. 1, 2).
The variations within the haplotypes of T. vaporariorum mtCOI partial gene was observed for groups made out by partitioning samples based on collected regions as Uttarakhand, Himachal Pradesh and Others (New Delhi and Tripura). The number of haplotypes, total number of variable sites, and haplotype diversity, and nucleotide diversity, average number of nucleotide differences, G+C content and total number of mutations were represented in Table 2. A total of 20 haplotypes were identified from the 32 sequences of our study. Haplotype 2 (H2) was found dominant by sharing 13 sequences where in which 11 of them belong to Uttarakhand and 2 of them belong to Himachal Pradesh (Table 1).
Nucleotide diversity of the Uttarakhand observed to be 0.00346 whereas in Himachal Pradesh it was 0.00233 and the group-others (one sequence each of New Delhi and Tripura) was 0.00349. In the same way, haplotype diversity observed higher in others (1.000) followed by Uttarakhand (0.843) and Himachal Pradesh (0.667) ( www.nature.com/scientificreports/ minimum spanning network analysis (Fig. 3). The color code for the groups was represented differently i.e., Red indicates Himachal Pradesh, Violet indicates Uttarakhand, Yellow indicates New Delhi whereas Green indicates Tripura sequence. The 32 samples were diversified into a total of 20 haplotypes that were networked distantly from each other. Haplotype 2 was dominant by sharing 13 sequences and placed in the center of the network. The New Delhi and Tripura sequences form two unique haplotypes i.e., H19 and H20 respectively. Haplotype 2 shares 11 sequences of Uttarakhand and 2 sequences of Himachal Pradesh whereas the remaining haplotypes are unique from Uttarakhand region. The results indicate the variation among collected individuals of greenhouse whiteflies.

Discussion
The polyphagous whitefly species, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae), is a highly destructive pest of cold climate greenhouses 21 . It has been reported to infest over 102 herbaceous plants belonging to 36 families across the world 22  Integrated pest management approaches that combine cultural, physical, biological, and chemical control methods have been found to be effective in controlling T. vaporariorum infestations 24 . In addition, the development of resistant plant varieties could also serve as an effective long-term management strategy 25 . It is also important to note that T. vaporariorum infestations have been reported in other regions with similar climatic conditions, such as parts of Europe and the United States 26,27 . Overall, our findings highlight the need for continued monitoring and research on T. vaporariorum infestations in the Uttarakhand state of Indian Himalayas and other regions with similar climatic conditions. In the present study, the Uttarakhand state located in Indian Himalayan region of India, was surveyed to detect diversity of T. vaporariorum populations. Whitefly survey using aspirator tools are widely used tools to collect whitefly for phylogeography study 28 . The molecular diagnostic technique deployed was convenient to identify whitefly populations and expanding our knowledge of T. vaporariorum occurrence and diversity in Uttarakhand state.
Molecular diversity techniques have been proven useful in unravelling diverse insect pest of agricultural importance including new species 29 , biotypes 30 , cryptic species 31 and haplotypes 32 . The presence of T.  In the construction of the tree, a total of 32 partial COI sequences obtained from the present study were combined with reference sequences from public databases and outgroup sequences (AY057182.1) retrieved from GenBank. Each sequence is labeled with a sample name corresponding to the samples collected and studied in this research. The presence of T. vaporariorum may have harmful impact on yield of protected crops grown in the regions due to agility in adaptiveness, colonization in newer habitats and development of insecticidal resistance 34,35 .
The mtCOI gene sequence amplification found useful for the detection of whitefly species. mtCOI gene have been used as potential marker in the past for discrimination of white fly species complex, evolutionary and genetic diversity study with accuracy and reliability [36][37][38] . The phylogenetic tree reconstructed by retrieving and aligning mtCOI sequences with the expansion of mtCOI supports the relatedness and validity of the molecular identification of collected T. vaporariorum.
Population genetic study using mtCOI suggest the high variation with presence of 20 haplotype in the T. vaporariorum populations. Most of the haplotype differs with single nucleotide mutations with their corresponding haplotypes except the haplotype number 1, 13, 15, 16. The result of this study gives fundamental knowledge on understanding of geographical distribution and ecology of T. vaporariorum in Uttarakhand. The host factor and ecological adaptability may be the reason for high abundance in different area 33 . However, the effect of several other factors such as altitudinal variation, temperature effect, crop cycle, rainfall pattern, wind directions, wind speed, dispersal through planting material and existence of natural population could be explored for future studies to get real scenario and cause of distribution. We have revealed here, the wide distribution pattern of T. vaporariorum on different host at different altitudes exhibiting uniformity in genetic level. The detection of the whitefly population in Uttarakhand state areas may help us to extend surveillance, monitoring and decision making about T. vaporariorum management initiative in the identified areas. Additionally, the present study attracts special attention to study the associated viruses being vectored by T. vaporariorum on different host plants.

Conclusion
Global distribution, polyphagous nature and virus vector potential of Greenhouse whitefly (GWF) Trialeurodes vaporariorum (Westwood) is a serious concern for farming community. The spread of GWF into new area may cause mayhem to the crops. Therefore, regular survey, identification, documentation on geospatial distribution of GWF is urgent need. Present study, suggest the frequent occurrence of GWF from Indian Himalayan states. Population genetic study using mtCOI suggest the high variation with presence of 20 haplotype in the T. vaporariorum populations, where haplotype 2 (H2) was found most dominant. The obtained result draws attention of researchers, policy makers and farmers for pest alertness, decision making research prioritization, development and adoption of sustainable management strategies of GWF to safeguard the crops.

Materials and methods
Survey sites and sample collection. A total of 32 greenhouse whitefly samples were collected from Uttarakhand, Himachal Pradesh, New Delhi and Tripura states during 2019-2021 from both protected cultivation systems as well open field conditions (Table 3). Among which, 27 samples were collected from Uttarakhand state located in Northern India, bordering the Tibet Autonomous Region of China in the north, Nepal in the east, whereas, 3 samples were collected from Himachal Pradesh (Fig. 4). Both These states are part of North western Indian Himalayan region. The remaining two reference samples, one each were collected from New Delhi and Tripura respectively . The field surveys were conducted throughout the year from 2019 to 2021. The selected sur- www.nature.com/scientificreports/ vey sites in the two states of Indian Himalayan region were at the elevation of 300-2400 m above mean sea level with annual mean rainfall ranging between 750 and 1600 mm and mean minimum and maximum temperature ranging between 4.6-8.9 °C and 23.9-29.8 °C, respectively (Fig. 4). The survey sites were chosen randomly and indecently, irrespective of host plants, thus including both protected cultivation system and open field conditions. The sampled sites had no record of any insecticide application which could affect the whitefly populations. The adult whiteflies were captured from each site with the help of a hand-held aspirator. The captured whiteflies were placed in 1.5 ml Eppendorf tube containing 70% ethanol, labelled with sampling site and date description. The collected insects were stored at − 20 °C until further investigation. Nearly, 100 insects collected from one site were considered as a sample and single individual randomly was chosen for molecular characterization of the species.
DNA extraction, PCR amplification and Sanger sequencing. The standard methodology of CTAB as described by Subbanna et al. was followed for extraction of genomic DNA from an individual whitefly specimen 40 . Each individual whitefly was properly rinsed in sterile distilled water, crushed and homogenized using a hand-held homogenizer (Sigma Aldrich). 120 μl of CTAB solution (20 parts of 1 M Tris-HCl, 8 parts of 0.5 M EDTA, 56 parts of 5 M NaCl and 4 parts of CTAB and the final volume makeup 1000 ml and the pH adjusted to 8. After this 0.4 parts of ß-mercaptaethanol were added along with 1 mg/ml of proteinase K was transferred to the tube and incubated in a water bath at 57 °C for 1-3 h. The contents were vortexed manually after every 20 min for thorough degradation of the tissues. The degraded material was treated twice with phenolchloroform-isoamyl solution (25:24:1) to extract the genomic DNA and ice-cold isopropyl alcohol was used to precipitate the DNA at − 20 °C for 30 min. The DNA pellet obtained after centrifugation at 10,000 rpm for 7 min was washed with 70% chilled ethyl alcohol to remove the excess salts and was suspended in 20 μl of TE buffer. DNase-free RNase A treatment was followed for 20 min at 37 °C to remove the RNA residues. Electrophoresis with 0.8% agarose gel was carried out to visualize intact genomic DNA. Furthermore, the DNA concentration were quantified in Nanodrop (NanoDrop™ 2000/2000c) subsequently used as template for PCR amplification. The segments of mtCOI gene were amplified using pair of universal primers (COX1: LC01490-"5′-GGT CAA CAA ATC ATA AAG ATA-3′" and HC02198 "5′-TAA ACT TCA GGG TGA CCA AAAAA-3′") with 710 bp product 19,41 . The PCR reaction programme was set using 25 µl reaction mixtures including 12.5 µl of ready to use PCR master mix (Promega M750A), 5.5 µl of nuclease free water, 1 µl each of forward and reverse primer and 5 µl of DNA template. The thermo-cycler programme was run with the following cycles; 94 °C for 5 min as initial denaturation followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 52 °C for 45 s, extension at 72 °C for 45 s and the final extension step for 10 min at 72 °C. The presence of amplified PCR product was visualized and confirmed in the gel documentation system (Alpha Image Analyzer, Alpha Innotech Corporation) by 1.2% agarose-EtBr 10 mg/ml gel electrophoresis with 2.5 μl PCR product. Gel elution columns (Sigma) were used for purification of the amplified products of the target gene. The purified products were sequenced directly by an automated DNA sequencer (ABI 377) following manufacturers guidelines for the Big Dye terminator kit (Applied Biosystems).
Similarity search, phylogenetic analysis and population genetics study. The raw reads of mtCOI gene sequence were assembled using Clustal Omega (1.2.2) multiple sequence alignment programme 42 . The sequences were exported to NCBI and BLASTn similarity search performed with default parameters. All the available whitefly sequences from databases were retrieved and aligned with ClustalW and bases were edited in BioEdit v7.2.5. Phylogeny was estimated with Mega X 43,44 following Kimura 2-parameter model and the maxi-